One of the goals of plant genetic engineering is to produce plants with agronomically desirable characteristics or traits. The proper expression of a desirable transgene in a transgenic plant is one way to achieve this goal. Elements having gene regulatory activity, i.e. regulatory elements such as promoters, leaders, introns and transcription termination regions, are polynucleotide molecules which play an integral part in the overall expression of genes in living cells. Isolated regulatory elements that function in plants are therefore useful for modifying plant phenotypes through the methods of genetic engineering.
Many regulatory elements are available and are useful for providing good overall gene expression. For example, constitutive promoters such as P-FMV, the promoter from the 35S transcript of the Figwort mosaic virus (U.S. Pat. No. 6,051,753, herein incorporated by reference); P-CaMV 35S, the promoter from the 35S RNA transcript of the Cauliflower mosaic virus (U.S. Pat. No. 5,530,196, herein incorporated by reference); P-Rice Actin 1, the promoter from the actin 1 gene of Oryza sativa (U.S. Pat. No. 5,641,876, herein incorporated by reference); and P—NOS, the promoter from the nopaline synthase gene of Agrobacterium tumefaciens are known to provide some level of gene expression in most or all of the tissues of a plant during most or all of the plant's lifespan. While previous work has provided a number of regulatory elements useful to affect gene expression in transgenic plants, there is still a great need for novel regulatory elements with beneficial expression characteristics. Many previously identified regulatory elements fail to provide the patterns or levels of expression required to fully realize the benefits of expression of selected genes in transgenic crop plants. One example of this is the need for regulatory elements capable of driving gene expression in different types of tissues.
Expression elements which are preferentially control gene expression in mesophyll or bundle sheath cells are particularly desirable. For example, such elements may be utilized to control agronomically important characteristics such as photosynthetic activity or yield enhancement. Promoters which have been shown to demonstrate such activity include the Zea mays fructose 1-6 bisphosphate aldolase (FDA) promoter (U.S. patent application Ser. No. 09/757,089, herein incorporated by reference), provided as SEQ ID NO: 3 and SEQ ID NO: 4, pyruvate orthophosphate dikinase (PPDK) promoter (U.S. Pat. No. 6,645,765, herein incorporated by reference), provided as SEQ ID NO: 2, and the ribulose bisphosphate carboxylase activase (RUA) promoter (U.S. patent application Ser. No. 11/514,704, herein incorporated by reference), provided as SEQ ID NO: 1. Another example is the need for elements other than promoters to provide alternate mechanisms for the regulation of gene expression. Additionally, different combinations of elements may result in promoters able to drive gene expression in different or additional plant cell types than any of the parent elements.
The genetic enhancement of plants and seeds provides significant benefits to society. For example, plants and seeds may be enhanced to have desirable agricultural, biosynthetic, commercial, chemical, insecticidal, industrial, nutritional, or pharmaceutical properties. Despite the availability of many molecular tools, however, the genetic modification of plants and seeds is often constrained by an insufficient or poorly localized expression of the engineered transgene.
Many intracellular processes may impact overall transgene expression, including transcription, translation, protein assembly and folding, methylation, phosphorylation, transport, and proteolysis. Intervention in one or more of these processes can increase the amount of transgene expression in genetically engineered plants and seeds. For example, raising the steady-state level of mRNA in the cytosol often yields an increased accumulation of transgene expression. Many factors may contribute to increasing the steady-state level of an mRNA in the cytosol, including the rate of transcription, promoter strength and other regulatory features of the promoter, efficiency of mRNA processing, and the overall stability of the mRNA.
It is of immense social, ecological and economic interests to develop plants that have enhanced nutrition, improved resistance to pests, and tolerance to harsh conditions such as drought. Thus, the identification of new genes, regulatory elements (e.g., promoters), etc. that function in various types of plants is useful in developing enhanced varieties of crops. Clearly, there exists a need in the art for new regulatory elements, such as promoters, leaders and enhancers, that are capable of expressing heterologous nucleic acid sequences in important crop species.
Chimeric Regulatory Elements
In addition to the utility of single gene expression elements, we have found that hybrid or chimeric expression elements derived from isolated regulatory elements from corn, particularly the promoter, enhancer and leader regulatory elements, provide enhanced expression patterns for an operably linked transgene in transgenic plants. It is known that some types of promoters may preferentially drive gene expression in specific tissues, such as leaf. Some promoters may additionally exhibit cell-specific gene expression, such as mesophyll cells or bundle sheath cells. We found that chimeric expression elements provides unexpected expanded expression patterns not seen with native gene regulatory elements. In particular, expression elements normally modulating gene expression in either mesophyll cells (for example, pyruvate orthophosphate dikinase or PPDK elements) or leaf bundle sheath cells (for example, fructose 1-6 bisphosphate aldolase or FDA elements, and ribulose bisphosphate carboxylase activase or RUA elements) may be combined in novel ways to provide gene expression regulation in both mesophyll and leaf bundle sheath tissues. Additional elements which may contribute to enhanced regulatory activity may include the HSP70 intron (U.S. Pat. No. 5,859,347, herein incorporated by reference).
The development of chimeric or hybrid expression elements to tailor the expression pattern of particular genes would be of great interest in the development of plants that exhibit agronomically desirable traits, preferably those related to insect resistance, disease resistance, stress tolerance, herbicide tolerance, or yield.